Antibodies comprising modified heavy constant regions

Abstract

Provided herein are heavy chain constant regions (referred to as “modified heavy chain constant regions”), or functionally equivalent fragments thereof, that (i) enhance biological properties of antibodies relative to the same antibodies in unmodified form or (ii) reduce effector function. An exemplary modified heavy chain constant region includes an IgG2 hinge and three constant domains (i.e., CH1, CH2, and CH3 domains), wherein one or more of the constant region domains are of a non-IgG2 isotype (e.g., IgG1, IgG3 or IgG4). The heavy chain constant region may comprise wildtype human IgG domain sequences, or variants of these sequences. Also provided herein are methods for enhancing certain biological properties of antibodies that comprise a non-IgG2 hinge, such as internalization, agonism and antagonism, wherein the method comprises replacing the non-IgG2 hinge of the antibody with an IgG2 hinge.

1 . An isolated antibody or fusion protein comprising a heavy chain constant domain of an IgG1 or IgG2 antibody or a portion thereof or comprising an amino acid sequence selected from SEQ ID NOs: 233 (IgG1fa), 240 (IgG2.3), 242 (IgG2.3G1), 246 (IgG1f), 255 (IgG2.5) and 257 (IgG2.5G1), wherein the heavy chain constant domain further comprises a substitution at one or more of amino acid residues L234, L235, P238, G237 and K322, wherein the antibody or fusion protein has (i) reduced binding to a low affinity Fc receptor; (ii) reduced binding to a high affinity Fc receptor; (iii) reduced binding to C1q; (iv) reduced ADCC; (v) reduced ADCP; and/or (vi) reduced CDC relative to the same antibody or fusion protein but without the amino acid substitution at one or more of amino acid residues L234, L235, P238, G237 and K322. 
     
     
         2 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain:
 is that of an IgG1 antibody or a portion thereof or comprises an amino acid sequence selected from SEQ ID NOs: 233 (IgG1fa), 242 (IgG2.3G1), 246 (IgG1f), and 257 (IgG2.5G1), and wherein the heavy chain constant domain further comprises a substitution at one or more of amino acid residues L234, L235, P238, G237 and K322; or   is that of an IgG2 antibody or a portion thereof or comprises an amino acid sequence selected from SEQ ID NOs: 240 (IgG2.3) or 255 (IgG2.5), and wherein the heavy chain constant domain further comprises a substitution at one or more of amino acid residues P238 and K322.   
     
     
         3 . The isolated antibody or fusion protein of  claim 1 , wherein the substitution at L234 is L234A or a conservative substitution thereof; the substitution at L235 is L235E or a conservative substitution thereof; the substitution at P238 is P238K or a conservative substitution thereof; the substitution at G237 is G237A or a conservative substitution thereof and the substitution at K322 is K322A or a conservative substitution thereof. 
     
     
         4 . The isolated antibody or fusion protein of  claim 1 , wherein the substitution at L234 is L234A; the substitution at L235 is L235E; the substitution at P238 is P238K; the substitution at G237 is G237A and the substitution at K322 is K322A. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises at least a portion of the hinge that is from an IgG1 antibody, and comprises a substitution at P238 or a conservative substitution thereof, and a substitution at L235E or a conservative substitution thereof. 
     
     
         8 . The isolated antibody or fusion protein of  claim 7 , wherein the heavy chain constant domain does not comprise any other mutation that reduces effector function. 
     
     
         9 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises at least a portion of the hinge that is from an IgG1 antibody, and comprises:
 (a) a substitution at P238 or a conservative substitution thereof, a substitution at L235E or a conservative substitution thereof and a substitution at K322A or a conservative substitution thereof;   (b) a substitution at P238 or a conservative substitution thereof and a substitution at K322A or a conservative substitution thereof;   (c) a substitution at P238 or a conservative substitution thereof, a substitution at L235E or a conservative substitution thereof; a substitution at K322A or a conservative substitution thereof and a substitution at L234 or a conservative substitution thereof;   (d) a substitution at P238 or a conservative substitution thereof, a substitution at L235E or a conservative substitution thereof; a substitution at K322A or a conservative substitution thereof; a substitution at L234 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (e) a substitution at P238 or a conservative substitution thereof, a substitution at L235E or a conservative substitution thereof; a substitution at K322A or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (f) a substitution at P238 or a conservative substitution thereof; a substitution at K322A or a conservative substitution thereof; a substitution at L234 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (g) a substitution at P238 or a conservative substitution thereof; a substitution at L234 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (h) a substitution at P238 or a conservative substitution thereof and a substitution at L234 or a conservative substitution thereof;   (i) a substitution at P238 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (j) a substitution at P238 or a conservative substitution thereof; a substitution at L235 or a conservative substitution thereof; a substitution at L234 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (k) a substitution at P238 or a conservative substitution thereof; a substitution at L235 or a conservative substitution thereof; and a substitution at L234 or a conservative substitution thereof;   (l) a substitution at P238 or a conservative substitution thereof; a substitution at L235 or a conservative substitution thereof; and a substitution at G237 or a conservative substitution thereof;   (m) a substitution at P238 or a conservative substitution thereof; a substitution at K322 or a conservative substitution thereof; a substitution at L234 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof;   (n) a substitution at P238 or a conservative substitution thereof; a substitution at K322 or a conservative substitution thereof; and a substitution at L234 or a conservative substitution thereof; or   (o) a substitution at P238 or a conservative substitution thereof; a substitution at K322 or a conservative substitution thereof and a substitution at G237 or a conservative substitution thereof.   
     
     
         10 - 38 . (canceled) 
     
     
         39 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises at least a portion of the hinge that is from an IgG1 antibody, and comprises one of the following substitutions or groups of subsitutions: P238K; P238K/L235E; P238K/L235E/K322A; P238K/K322A; P238K/L234A/G237A; P238K/G237A; P238K/L234A; P238K/L235E/L234A/G237A; P238K/L235E/G237A; P238K/L235E/L234A; P238K/K322/L234A/G237A; P238K/K322/G237A and P238K/K322/L234A. 
     
     
         40 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises one of the following substitutions or groups of subsitutions: P238K and P238K/K322A. 
     
     
         41 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to that of a wildtype IgG1 or IgG2 or one of SEQ ID NOs: 233 (IgG1fa), 240 (IgG2.3), 242 (IgG2.3G1), 246 (IgG1f), 255 (IgG2.5) and 257 (IgG2.5G1) or an allotypic variant of any of these, except for the one or more amino acid substitutions recited in one of claims  186 - 225 . 
     
     
         42 . (canceled) 
     
     
         43 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain comprises an amino acid sequence selected from SEQ ID NOs: 235-239, 241, 243, 244, 245, 252-254, 256 and 259-262. 
     
     
         44 . The isolated antibody or fusion protein of  claim 1 , wherein P238 is P238R or P238H. 
     
     
         45 . The isolated antibody or fusion protein of  claim 1 , wherein L235 is L235D. 
     
     
         46 . The isolated antibody or fusion protein of  claim 1 , wherein K322 is K322V, K322L, K322I, K322P, K322F or K322M. 
     
     
         47 . The isolated antibody or fusion protein of  claim 1 , wherein the heavy chain constant domain does not comprise the C-terminal lysine.